Multidrug resistance relates to resistance of tumor cells to a whole range of chemotherapy drugs with different structures and cellular targets [Leonard et al., The Oncologist 8:411-424 (2003)]. The phenomenon of multi drug resistance (MDR) is a well known problem in oncology and thus warrants profound consideration in considering various cancer treatments. One of the underlying molecular rationales for MDR is the up-regulation of a family of MDR transporter proteins that cause chemotherapy resistance in cancer by actively extruding a large variety of therapeutic compounds from the malignant cells. MDR transporters belong to the evolutionarily conserved family of the ATP binding cassette (ABC) proteins, and are presented in practically all living organisms from prokaryotes to mammals [Gottesman et al., Nat Rev Cancer 2:48-58 (2002)]. ABC transporters play an important protective function against toxic compounds in a variety of cells and tissues, especially in secretory organs, at the sites of absorption, and at blood-tissue barriers. The three major multidrug resistance ABC proteins are MDR1 (including P-glycoprotein, ABCB1), multidrug resistance associated protein 1 (including MRP1, ABCC1) and ABCG2 (including placenta-specific ABC transporter, ABCP/breast cancer resistance protein, BCRP/mitoxantrone resistance protein and MXR). MDR1 and MRP1 can transport a large variety of hydrophobic drugs, and MRP1 can also extrude anionic drugs or drug conjugates. Other members of the MRP/ABCC family have also been indicated to be involved in cancer multidrug resistance (for details, see [Haimeur et al., Curr Drug Metab 5:21-53 (2004)]). The transport properties of ABCG2 are overlapping with those of both MDR1 and the MRP type proteins; thus these three proteins form a special network in chemo-defense mechanisms.
Because of a significant role that ABC transporters play in cancer multidrug resistance and the body's protection against xenobiotics, sensitive and specific quantitative assays are required for the detection of the activity of these proteins. High throughput assay systems are also required to screen for potential transporter-interacting partners. The estimation of the activity of ABC transporter is not easily achieved by the routinely available classical non-functional methods, such as Northern blotting, RNase protection, RNA in situ hybridization, RT PCR or immunostaining MDR-ABC protein expression is often not correlated with mRNA levels or is often below the detection threshold, as relatively few active transporter molecules may cause major alterations in drug transport. Additionally, functional activity of ABC transporters may not correlate with their expression levels determined by the foregoing methods.
The ability of ABC transporters to actively transport compounds against the concentration gradient across the cell membrane has allowed the development of a number of functional assays to measure the level and function of transporter protein. Lipophilic dyes are capable of diffusing across cell membranes. Upon the loading of the cells with the dye(s), the resulting fluorescence intensity will depend on the activity of ABC transporters; the cells with highly active transporters will demonstrate lower fluorescence because of the increased efflux of the dye/substrate. The functions of MDR-ABC transporters have been characterized by measuring the cellular uptake, efflux, or steady-state distribution of a number of fluorescent substrates (summarized in Table 1, FIG. 1A-1C) using flow cytometry, fluorescent microscopy or fluorimetry. Substrate specificities of Pgp, MRP and BCRP are distinct, but also overlapping [Litman et al., Cell Mol Life Sci. 58:931-959 (2001)], as shown in Table 1, FIG. 1A-1C.
Several drawbacks have been noted relating to the sensitivity of most fluorophores used in this application because of protein binding, sequestration, or changes in fluorescence due to the intracellular milieu such as pH or free calcium levels [Hollo et al., Biochem Piophys Acta 1191:384-388 (1994)]. To increase sensitivity of the method, hydrophobic ester derivatives, such as AM esters of the fluorescent dyes have been used. These cell permeable derivatives of fluorescent dyes are also actively exported from cells by MDR proteins. When these ester derivatives reach the cytosol, however, intracellular esterases cleave the ester groups so that the dyes are now impermeable and require active transport. The resulting free dye can now be actively exported by MDR proteins in the cells [Homolya et al., J Biol Chem. 268:21493-21496 (1993); Liminga et al., Exp Cell Res. 212:291-296 (1994)]. Some of the dyes are only fluorescent after AM hydrolysis.
Clinicians are especially interested in identifying the drug resistance profile, and the substrate specificity and drug extrusion activity of the various multi-drug resistance proteins expressed in a given tumor sample. The demonstration of the transport activity of various multi-drug resistance proteins in the plasma membrane requires a sensitive and reproducible in vitro multi-drug resistance assay. To date, BCRP has gained prominence as one of the three major ATP-binding cassette (ABC) membrane efflux transporters alongside P-gp and MRP, conferring drug resistance in cancer and inflammation chemotherapies [Litman et al., J Cell Sci. 113:2011-2021 (2000); van der Heijden et al., Arthritis Rheum. 60:669-677 (2009); Koshiba et al., Xenobiotica 38:863-868 (2008)]. Besides being present in drug-resistant cancer and T-cells, BCRP is also endogenously expressed at a high level in human placenta and to a lesser extent in liver, small intestine and colon, ovary, veins, capillaries, kidney, adrenal, and lung, with little to no expression in brain, heart, stomach, prostate, spleen, and cervix [Doyle et al., Oncogene 22(47):7340-7358 (2003)]. It has been demonstrated that calcein AM is useful for the quantitative functional analysis of the presence of active ABCB and ABCC but not ABCG transporters in cells [Litman et al., J Cell Sci. 113:2011-2021 (2000)]. Thus, it is important to have a simple, reproducible and sensitive method to determine activity of all three types of ABC transporters in live cells.
To detect functional activity of drug efflux pumps, two assays using fluorescent dyes are commonly employed: one measures cellular dye accumulation and the other measures dye retention. The accumulation assay measures dye uptake in the presence or absence of known pump modulators. In the retention assay, the cells are loaded with the substrate in the absence of any reversal agent, washed, and then further incubated without dye but in the presence of reversing agents to allow time for the substrate to be transported out of the cell by a drug efflux pump. The distinction between accumulation and retention is necessary when evaluating cells for MDR phenotypes, as substrates act differently under different experimental conditions. Accumulation/retention assays offer high throughput, generic readouts (increase in fluorescence intensity), and are readily automated. These assays are not designed, however, to distinguish Pgp substrates from inhibitors [Tiberghien, F. and Loor, F., Anticancer Drugs 7:568-578 (1996)] and do not directly measure transport.
The cell-based bidirectional permeability assay (or monolayer efflux assay) is currently accepted as the “method of choice” for determining the P-gp inhibition potential of test compounds in drug discovery laboratories and across the industry [Horio et al., J Biol Chem. 264:14880-14884 (1989); Horio et al., Proc Natl Acad Sci USA. 85:3580-3584 (1988); Polli et al., J Pharmacol Exp Ther. 299:620-628 (2001); Rautio et al., Drug Metab Dispos 34(5) 786-792 (2006)]. The FDA guidance document [Drug Guidance for Industry, http://www.fda.gov/cder/guidance/6695dft.pdf, (2006)] also recommends conducting bidirectional permeability studies using Caco-2 cells or other cell lines (e.g., MDCK, LLC-PK1 etc.), either wild-type or transfected (with P-gp or other MDR proteins), to determine the P-gp inhibition potential of test compounds [Zhang et al., Xenobiotica 38:709-724 (2008)]. The experimental protocol for this assay is well established and quite uniform across different laboratories. Typically, bidirectional permeability of a well accepted MDR substrate is assessed alone and in the presence of a single concentration of test compound to estimate the inhibition potential.
This type of the assay is generally conducted for studying in vitro drug interactions with membrane transporter proteins that belong to two major superfamilies—ABC transporters [rev. in Schinkel, A. H. and Jonker, J. W., Adv Drug Deli Rev. 55:3-29 (2003)] and solute carrier (SLC) transporters [rev. in Hediger et al., Pflugers Arch. 447:465-468 (2004)]. Many pharmaceutical agents and toxins are transported by multispecific SLC transporters for organic anions (e.g., OATP-SLCO/SLC21 and OAT-SLC22A1-3) and organic cations (OCT-SLC 22A4-5). These solute carriers, often called “uptake transporters,” include a large variety of related membrane proteins, and in many cases possess overlapping substrate profiles with the MDR-ABC proteins [Marzolini et al., Pharmacogenomics 5:273-282 (2004); Miyazaki et al., Trends Pharmacol Sci 25:654-662 (2004)]. Some of these transporters perform obligatory exchange of organic compounds (e.g., OAT3), while in others transport is modulated and/or driven by monovalent ions and the membrane potential (e.g., OCTN). Moreover, there is a coordinated expression pattern for various SLC and ABC transporters in the liver, kidney, and BBB, which may govern the overall vectorial transport of many compounds that are mutual substrates of MDR-ABC and SLC proteins [Kusuhara, H. and Sugiyama, Y., Neuro Rx 2:73-85 (2005)].
Transepithelial transport depends on the polar distribution of proteins and lipids in the plasma membrane of epithelial cells. Typically, one plasma membrane contains a transporter that allows a specific ligand to enter the epithelial cells, and the plasma membrane on the opposite side of the cell contains a transporter that functions to export the ligand from the cell, resulting in net transepithelial transport of the ligand from one side of the epithelium to the other. In general, the substrates for transport by MDR proteins are highly lipophilic, and so enter epithelial cells freely from both surfaces. MDR proteins in the apical plasma membrane could expel substrates across the apical plasma membrane. Thus, ligand entering the cell from the basolateral surface is likely to leave the cell across the apical plasma membrane, whereas ligand entering the cell across the apical plasma membrane is likely to leave the cell across the same plasma membrane, with the result that basolateral to apical flux exceeds apical to basolateral flux. The net effect would be secretion of cytotoxic drugs by each of the epithelia listed above.
The monolayer efflux assay, where the ratio of basolateral-to-apical (B→A) permeability versus apical-to-basolateral (A→B) permeability is compared with a value of 1, is regarded as the standard for identifying MDR substrates because this assay measures efflux in the most direct manner [Polli et al., J Pharmacol Exp Ther 299:620-628 (2001)]. Due to concentration-dependent inhibition of active efflux on the apical side by MDR inhibitors, the B→A permeability decreases whilst A→B permeability increases with ratio approaching unity as the dose of inhibitor increases. The affinity of inhibitor to MDR protein(s) may be studied by calculating the active flux that can be obtained from the B→A fluxes in the absence and presence of MDR inhibitors Inhibition potency, determined by inhibitor concentration-dependent transport assay, is usually represented as an IC50 value, the concentration that gives 50% of maximum inhibition of a known P-gp substrate. Specificity of the inhibitor to P-gp may be determined by competitive assays, which involve a transport assay of a known substrate in the presence of known inhibitors (specific to various transporters) with and without compounds under test. Thus, appropriate design of competitive inhibition assays using known selected substrates and modulators will show the specificity of the inhibitors towards the efflux pump.
Bacterial multidrug efflux systems are a serious problem in the pharmacological treatment of patients with infectious diseases since the substrate spectra of many multidrug transporters include clinically relevant antibiotics [Putman et al., Microbiol Mol Biol Rev. 64(4):672-693 (2000)]. Multidrug resistant pumps operate via an active efflux mechanism and are typically ATP- or PMF-(proton motive force) dependent. Multidrug transporters are associated with both intrinsic and acquired resistance to antibiotics. For example, homologous to human P-gp ABC-type multidrug transporter LmrA of L. lactis displays very broad antibiotic specificity, demonstrating that ATP-dependent multidrug transporters can seriously affect the efficacy of many antibiotics [van Veen et al., Proc Natl Acad Sci ISA 93:10668-10672 (1996); van Veen et al., Nature 391(6664):291-295 (1998)]. Dyes such as acriflavine, ethidium bromide, rhodamine 6G and pyronin Y are used as substrates for many bacterial efflux pumps and their susceptibility in the presence or absence of efflux pump inhibitors has been used as a screen for the presence of efflux-related resistance mechanisms [DeMarco et al., Antimicrob Agents Chemother. 51:3235-3239 (2007)]. The bacterial multidrug efflux systems transport similar drugs and are sensitive to similar inhibitors as the mammalian multidrug transporter, P-gp [Neyfakh et al., Proc Natl Acad Sci USA. 88:4781-4785 (1991)].
There remains a need for a general non-toxic MDR probe that a) will recognize all three major ABC transporter's types in live cells (unlike the most common MDR probe Calcein AM and other calcium indicator dyes such as Fluo-3 and Fluo-4); b) will be brighter (e.g. possess higher quantum yield and molar extinction coefficient) and more sensitive than common general MDR probes, such as doxorubicin and mitoxantrone; c) could be used in all types of the efflux assays discussed above; d) will require few processing steps and provide a reproducible protocol; and e) will be compatible with other common fluorescent dyes used in flow cytometry and diverse fluorescent proteins expressed in cells.